MicroRNA Regulation of NMNAT-Mediated Neuroprotection Against Peripheral Neuropathy and Chronic Pain PROJECT SUMMARY Peripheral neuropathy and neuropathy pain can be caused by a myriad of genetic and environment factors as well as therapeutic or recreational drug use. In particular, chemotherapy-induced peripheral neuropathy (CIPN) is the major dose-limiting neurotoxic side effect of standard chemotherapy regiments. Over 68% of cancer patients experience neuropathic symptoms after chemotherapy, and that contributes to a significant percent of the population that suffer from chronic pain and have to resort to opioid use. Currently there are no effective treatments available, largely due to a lack of understanding of the in vivo mechanisms of CIPN and related peripheral neuropathy. Recently, we have optimized a model of peripheral neuropathy using Drosophila larvae that recapitulates salient behavior and neuronal morphological aspects of chemotherapy- induced sensory dysfunction. Our preliminary work using this model has uncovered a new mechanism underlying peripheral neuropathy, and identified a neuroprotective protein NMNAT with promising potential for mitigating neuropathic pain. The ultimate goal of our research is to uncover the endogenous mechanisms underlying peripheral neuropathy and to identify neuroprotective mechanisms and potential targets that facilitate the development of new therapeutic agents against CIPN and related neuropathic pain. Our previous work on neuronal maintenance and protection has identified NMNAT (nicotinamide mononucleotide adenylyl transferase), the last enzyme in NAD+ synthesis pathway, as an essential gene that maintains neuronal structural and functional integrity. Extensive mechanistic studies from our lab and others have found NMNAT proteins in Drosophila and mammals to be among the most robust and versatile neuroprotective factors, and a positive correlation between NMNAT expression levels and the neuronal self- protective capacity. Excitingly, from a compound screen, several natural compounds were identified to upregulate NMNAT transcription, and we have collected intriguing preliminary results suggesting that the expression of NMNAT is regulated by microRNAs. We hypothesize that regulation of NMNAT RNA expression by natural compounds and microRNAs at the steps of transcription, pre-mRNA splicing, and mRNA stability allows rapid and dynamic shifting between NMNAT mediated NAD+ metabolism and neuronal resilience, and confers protection in sensory neurons against peripheral neuropathy. In this application we outline experiments to (1) identify microRNAs that regulate nociceptive hypersensitivity, (2) identify and characterize the molecular pharmacology of natural compounds in regulating NMNAT expression, and (3) modulate NMNAT transcriptional regulation to enhance neuroprotection against peripheral neuropathy. Within the network of convergent pathways responding to chemotherapy induced peripheral neuropathy, understanding the regulation of NMNAT in both NAD+-metabolism and enhancing neuronal homeostasis will facilitate discovery of neuroprotective strategies in peripheral neuropathy and neuropathic pain.